Quinolones are some of the most frequently used, broad-spectrum antimicrobial agents. Resistance to these drugs has become a critical public health problem. Mutations in the drug target topoisomerases that result in drug resistance are well documented. In addition, overproduction of the multidrug efflux pump AcrAB has been reported to result in quinolone resistance in clinical isolates of E. coli. Our long-range goal is to determine how bacteria respond to exposure to quinolone agents and to use this knowledge to design more effective treatments. A goal of the present proposal is to identify the genetic alterations that lead to quinolone resistance in clinical E. coli strains and to determine the interrelationship between levels and frequency of quinolone resistance, the mutations, and other patient covariates. The central hypothesis is that following quinolone treatment, mutations conferring resistance occur additively, beginning with mutations in the topoisomerases and ultimately including overproduction of AcrAB and that the most resistant isolates contain additional mutations, including another multidrug efflux pump. Preliminary data support these hypotheses. Overproduction of any multidrug efflux pump has far reaching therapeutic consequences; antimicrobial agents from multiple different categories, in addition to the quinolones, would be ineffective against these bacteria. With the combined basic, clinical, statistical, genomic, and bioinformatic expertise of the investigators and the size of our patient population, we are uniquely poised to carry out the following specific aims: (1) Identify and categorize genetic alterations that cause quinolone resistance in clinical isolates. We will use high through-put methods to detect mutations in the genes that encode quinolone resistance. Statistical methods will be used to analyze potential interrelationships between the mutations. (2) Perform a prospective analysis of patient data. We will: (a) use the high-throughput methods developed in specific aim 1 to determine the genetic alterations occurring in E. coli isolated from patients hospitalized in the Texas Medical Center compared to isolates from various consortia around the world; (b) perform genome typing; (c) analyze these data with respect to demographic and clinical data for the patients to determine the probable causes of quinolone resistance. Until we have a better understanding of the mechanisms used by bacteria to cope with drug pressure, we cannot design better inhibitors or control antimicrobial resistant infections.